Xylans are heteropolysaccharides that form the major part of the hemicellulose present in the plant biomass. The backbone of these polysaccharides is a chain of β 1-4 linked xylopyranosyl residues. Many different side groups could bind to these residues like acetyl, arabinosyl and glucuronosyl residues. Phenolic compounds such as ferulic or hydroxycinnamic acids are also involved through ester binding in the cross linking of the xylan chains or in the linkage between xylan and lignin chains for example.
Endoxylanases (or endo-β-1,4-xylanases) hydrolyse specifically the backbone of the hemicellulose. In some cases, the side groups may mask the main chain by steric hindrance. Different xylanases activities have been described. Their specificity towards their substrate varies from one to each other. Some are more active on insoluble arabinoxylans. The length of the oligomers produced also depends on the type of xylanase considered.
Glycoside hydrolases (formerly known as cellulase family D) have been classified in 91 families (http://afmb.cnrs-mrs.fr/CAZY/) based on sequence homologies, structural and mechanistic features. Because the fold of proteins is better conserved than their sequences, some of the families can be grouped in ‘clans’ (Henrissat B. 1991, Biochem. J. Vol. 280, p 309). Endo-beta-1,4-xylanases are generally classified in families 10 (formerly family F) and 11 (formerly family G) and are found to frequently have an inverse relationship between their pI and molecular weight. The family 10 xylanases (EXs10) are larger, more complex, while the family 11 xylanases (EXs11) are smaller. Moreover a significant difference in the structure and the catalytic properties of both families exists. EXs10 present an (α/β)8 barrel fold, have about 40% of α-helix structures and belong to clan GH-A (Dominguez et al., 1995, Nat. Struct. Biol. Vol. 2, p 569) while EXs11 exhibit a β-jelly fold conformation, have about 3-5% of α-helix structures and belong to clan GH-C (Törrönen et al., 1994, EMBO J. Vol. 13, p 2498). EXs10 have a smaller substrate binding site and a lower substrate specificity, have frequently endoglucanase activity and produce smaller oligosaccharides compared to EXs11, which have a higher affinity for unsubstituted xylan (Biely et al., 1997, J. Biotechnol. Vol. 57, p 151). All xylanases of both families characterized to date retain the anomeric configuration of the glycosidic oxygen following hydrolysis in which two glutamates function as the catalytic residues (Jeffries, 1997, Curr. Opin. Biotechnol. Vol. 7, p 337).
Xylanases are used in various industrial areas such as the pulp, paper, feed and bakery industries. Other applications lie in the juice and beer sectors. Xylanases could also be used in the wheat separation process. The observed technological effects are, among others, improved bleachability of the pulp, decreased viscosity of the feed or changes in dough characteristics.
The use of xylanases (also called hemicellulases or pentosanases) in baking is well known since a number of years. These dough-conditioning enzymes can improve the dough machinability and stability as well as the oven-spring and the crumb structure. Other effects of the enzymes are a larger loaf volume and a softer crumb.
The mechanism of action of xylanases in bread preparation is still not clearly elucidated. There are about 3 to 4% pentosans in wheat flour. These pentosans can absorb large amounts of water (up to 30%). This water absorption contributes to the properties of the dough as well as to the quality of the final product. Partial hydrolysis of pentosans by pentosanases into water soluble short chain oligosaccharides increases the water binding capacity. There is also a strong interaction of the pentosans with the gluten fraction of the flour to form a network. Pentosanases may help to relax this strong and rigid network and therefore allowing the carbon dioxide formed by the yeast to better expand the dough.
Many types of hemicellulase preparations have been used as baking active-ingredients and are commercially available. They are produced by microbial fermentation using various microorganisms as enzyme sources. Some of these enzymes are also produced by genetically modified microorganisms. All documented uses of xylanases having a positive effect on the specific volume of baked goods, relate to xylanases belonging to glycoside hydrolases Family 10 or Family 11 as defined previously.
Examples of xylanases for baking are the xylanases from Bacillus subtilis and Aspergillus niger. 
There is a variety of methods to evaluate the xylanase activity in an enzyme preparation. Examples of such methods of xylanase activity determination are the measure of the release of reducing sugar from xylan (Miller G. L. 1959, Anal. Chem. Vol. 31, p 426) or the measure of the release of coloured compounds from modified substrates (for examples AzoWAX or Xylazyme AX from Megazyme). However no direct correlation could be found between the xylanolytic activity found in the various enzyme preparations and the effect in baking. Dose-related results can be observed to a certain extend for a single enzyme but the same dosage for two xylanases of different origins does not give the same result in the dough or in the bread. Several reasons could explain this: the differences in substrate specificity, the differences in temperature and pH optimum, . . . .
It is of great interest to develop new enzyme preparations, such as bread improver compositions or agents, with new or improved properties. One of these properties could be that the xylanase fraction is as small as possible (in terms of weight of enzymes needed to obtain a particular result in baking).
A xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis has been recently described (Collins, T. et al. 2002. A novel Family 8 xylanase: functional and physico-chemical characterisation. J. Biol. Chem. Vol. 277-38, p 35133; Collins, T. et al. 2003, Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. J. Mol. Biol. Vol. 328, p 419; Van Petegem F. et al., 2002. Crystallization and preliminary X-ray analysis of a xylanase from the psychrophile Pseudoalteromonas haloplanktis. Acta Crystallogr D Biol Crystallogr. Vol. 58(Pt 9), p 1494-6). This enzyme is a typical psychrophilic enzyme and presents a high catalytic activity at low temperature. It is not homologous to family 10 or 11 xylanases but has 20 to 30% identity with glycoside hydrolases family 8 (formerly family D) members, a family that comprises mainly endoglucanases but also lichenases and chitosanases. Furthermore, a FingerPRINTScan against PRINTS using the InterPro Scan search program (Zdobnov and Apweiler, 2001, Bioinformatics Vol. 17, p 847) indicated that the isolated sequence contained the glycosyl hydrolases family 8 fingerprint and family 8 residues that are strictly conserved in 20 family 8 enzymes analyzed.
Conversely to EXs10 and EXs11, this family 8 xylanase (EXs8) has both high pH and high molecular weight. Structural and catalytic properties are different to those of both EXs10 and EXs11. EXs8 present an (α/α)6 barrel fold with 13 α-helices and 13 β-strands and belong to clan GH-M (Van Petegem et al., 2003, The structure of a cold-adapted family 8 xylanase at 1.3 A resolution. Structural adaptations to cold and investigation of the active site. J. Biol. Chem. Vol. 278(9), p 7531-9). These enzymes have no endoglucanase, chitosanase or licheninase activity and appear to be functionally similar to EXs11, being more active on long chain xylo-oligosaccharides.
In contrast to other known EXs10 and EXs11 that retain the configuration, family 8 glyco-hydrolases (Fierobe et al., 1993. Eur. J. Biochem. Vol. 217, p 557; http://afmb.cnrs-mrs.fr/CAZY/) tend to hydrolyse the substrate with inversion of the anomeric configuration of the glycosidic oxygen following hydrolysis in which one glutamate and one aspartate function as the catalytic residues. This has been shown for example for the psychrophilic xylanase from Pseudoalteromonas haloplanktis (Van Petegem et al., 2003, J. Biol. Chem., Vol. 278(9), p 7531-9; Collins, T. et al. 2002. A novel family 8 xylanase: functional and physico-chemical characterization. J. Biol. Chem. Vol. 277(38), p 35133). Other xylanases belonging to the glycoside hydrolases family 8 have been already described (Yoon, K-H., 1998, Molecular cloning of a Bacillus sp. KK-1 xylanase gene and characterization of the gene product. Biochem. Mol. Biol. International. 45(2), p. 337; Bacillus halodurans C-125 xylanase Y GenBank/GenPept™ accession code BAB05824). These xylanases show sequence homologies between them as well as with the Pseudoalteromonas haloplanktis xylanase as described by Collins et al (2002, see above).
The list of the enzymes belonging to the glycoside hydrolases family 8 is regularly updated (http://afmb.cnrs-mrs.fr/CAZY/).